This invention relates to novel semi-synthetic antifungal compounds which are prepared by the acylation of the cyclic peptide nucleus produced by the enzymatic deacylation of antibiotic S31794/F-1.
Antibiotic S31794/F-1 is an antifungal cyclic peptide having the formula: ##STR3## wherein R is the myristoyl group. Throughout this application, the cyclic peptide formulas, such as formula I, assume that the amino acids represented are in the L-configuration.
Antibiotic S31794/F-1, which is disclosed in German Offenlegungschrift No. 2,628,965 and U.S. Pat. No. 4,173,629, is produced by Acrophialophora limonispora nov. spec. Dreyfuss et Muller NRRL 8095. S31794/F-1 has the following characteristics: m.p. 178.degree.-180.degree. C. (dec.) (amorphous) or 181.degree.-183.degree. C. (dec.) (crystalline); [.alpha.].sub.D.sup.20 -24.degree. (c 0.5, CH.sub.3 OH) or +37.degree. (c 0.5, pyridine) (crystalline); UV absorption maxima in methanol at 194 nm (E.sub.1 cm.sup.1% =807), 225 nm (shoulder) E.sub.1 cm.sup.1% =132), 276 nm (E.sub.1 cm.sup.1% =12.8), 284 nm (shoulder) E.sub.1 cm.sup.1% =10.5); .sup.13 C-NMR spectrum in deuteromethanol (190 mg in 1.5 ml deuteromethanol, tetramethylsilane as internal standard) with the following characteristics (crystalline):
______________________________________ PPM PPM PPM ______________________________________ 176.2 75.5 51.2 175.0 74.0 39.7 173.7 71.0 38.8 172.6 70.5 36.6 172.0 69.7 34.8 171.8 68.0 32.8 171.7 62.2 30.6 168.6 58.3 26.7 157.7 57.0 23.5 132.5 56.2 19.7 129.0 55.4 14.3 115.9 52.9 11.1 76.6 ______________________________________
an approximate elemental analysis (after drying crystalline material for two hours in a high vacuum at 100.degree. C.) as follows: 55.5-56.5 percent carbon, 7.5-7.7 percent hydrogen, 10.5-10.8 percent nitrogen and 25.5-26.0 percent oxygen; is readily soluble in methanol, ethanol, pyridine, dimethyl sulfoxide and poorly soluble in water, chloroform, ethyl acetate, diethyl ether, benzene and hexane; and has antifungal activity, especially against Candida albicans.
Antibiotic S31794/F-1 is prepared by submerged aerobic cultivation of Acrophialophora limonispora NRRL 8095 as described in Examples 6, 7 and 8. This microorganism is a part of the permanent culture collection of the Northern Regional Research Center, U.S. Department of Agriculture, Agricultural Research Culture Collection, North Central Region, Peoria, Ill. 61604, from which it is available to the public under the designated NRRL number.
Antibiotic S31794/F-1 has antifungal activity, particularly against Candida strains such as Candida albicans. Thus, production and isolation of the antibiotic can be monitored by bioautography using a Candida species such as Candida albicans.
In the antibiotic S31794/F-1 molecule (Formula I), the myristoyl side chain (R) is attached at the cyclic peptide nucleus at the .alpha.-amino group of the dihydroxyornithine residue. Surprisingly, it has been found that the myristoyl side chain can be cleaved from the nucleus by an enzyme without affecting the chemical integrity of the nucleus. The enzyme employed to effect the deacylation reaction is produced by a microorganism of the family Actinoplanaceae, preferably the microorganism Actinoplanes utahensis NRRL 12052, or a variant thereof. To accomplish deacylation, antibiotic S31794/F-1 is added to a culture of the microorganism and the culture is allowed to incubate with the substrate until the deacylation is substantially complete. The cyclic nucleus thereby obtained is separated from the fermentation broth by methods known in the art. Unlike antibiotic S31794/F-1, the cyclic nucleus (lacking the linoleoyl side chain) is substantially devoid of antifungal activity.
The cyclic nucleus afforded by the aforedescribed enzymatic deacylation of antibiotic S31794/F-1 is depicted in Formula II. ##STR4##
S31794/F-1 nucleus has an empirical formula of C.sub.35 H.sub.52 N.sub.8 O.sub.16 and a molecular weight of 840.87.
Removal of the side chain group affords a free primary .alpha.-amino group in the dihydroxyornithine residue of the cyclic peptide. For convenience, the compound having the structure given in Formula II will be referred to herein as "S31794/F-1 nucleus." As will be apparent to those skilled in the art, S31794/F-1 nucleus can be obtained either in the form of the free amine or of the acid addition salt. Although any suitable acid addition salt may be employed, those which are non-toxic and pharmaceutically acceptable are preferred.
The method of preparing S31794/F-1 nucleus from antibiotic S31794/F-1 by means of fermentation using Actinoplanes utahensis NRRL 12052 is described in the co-pending application of Bernard J. Abbott and David S. Fukuda, entitled "S 31794/F-1 NUCLEUS", Ser. No. 103,150, filed herewith this even date, which is a continuation-in-part of application Ser. No. 103,313, filed Dec. 13, 1979, the full disclosure of which is incorporated herein by reference. Example 5 herein, illustrates the preparation of S31794/F-1 nucleus by fermentation using antibiotic S31974/F-1 as the substrate and Actinoplanes utahensis NRRL 12052 as the microorganism.
The enzyme produced by Actinoplanes utahensis NRRL 12052 may be the same enzyme which has been used to deacylate penicillins (see Walter J. Kleinschmidt, Walter E. Wright, Frederick W. Kavanagh, and William M. Stark, U.S. Pat. No. 3,150,059, issued Sept. 22, 1964).
Cultures of representative species of Actinoplanaceae are available to the public from the Northern Regional Research Laboratory under the following accession numbers:
______________________________________ Actinoplanes utahensis NRRL 12052 Actinoplanes missouriensis NRRL 12053 Actinoplanes sp. NRRL 8122 Actinoplanes sp. NRRL 12065 Streptosporangium roseum var. hollandensis NRRL 12064 ______________________________________
The effectiveness of any given strain of microorganism within the family Actinoplanaceae for carrying out the deacylation of this invention is determined by the following procedure. A suitable growth medium is inoculated with the microorganism. The culture is incubated at about 28.degree. C. for two or three days on a rotary shaker. One of the substrate antibiotics is then added to the culture. The pH of the fermentation medium is maintained at about pH 6.5. The culture is monitored for activity using a Candida albicans assay. Loss of antibiotic activity is an indication that the microorganism produces the requisite enzyme for deacylation. This must be verified, however, using one of the following methods: (1) analysis by HPLC for presence of the intact nucleus; or (2) re-acylation with an appropriate side chain (e.g. linoleoyl, stearoyl, or palmitoyl) to restore activity.